1. Field of the Invention
This invention relates to the identification of polypeptides which have been separated on the same gel, typically from polyacrylamide gel electrophoresis (PAGE) and to a kit for use in the method. It is especially useful in proteomics (the large scale identification and characterisation of proteins).
2. Description of the Related Art
In proteomics, massively parallel protein identification and characterisation techniques are required. The identification of proteins or other polypeptides merely by PAGE, even using two-dimensional gels (2D-PAGE), is laborious and often uncertain. Many different methods have been developed to identify and partially characterise proteins from complex biological samples. Some of them use Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) techniques to analyse peptide "fingerprints" produced by fragmenting the proteins with enzymes. Several software programs have been developed to compare mass spectra of the peptides obtained from MALDI-TOF experiments with theoretical spectra from proteins. The subject has been reviewed by Kussmann and Roepstorff [1]. These authors noted three ways in which proteins separated by gel electrophoresis could be digested with enzymes to yield fragment peptides:
1. The digestion can be carried out in a plug of excised gel and the peptides recovered by elution. This is the authors' own preference. PA0 2. The protein can be first electroleluted from an excised gel plug and then digested in solution. PA0 3. The protein can be electroblotted onto a membrane and subsequently digested on the membrane. PA0 a) providing adjacent to the gel at least one hydrophilic membrane on which is immobilised at least one reagent capable of cleaving a polypeptide; PA0 b) providing a hydrophobic collection layer suitable for receiving thereon fragments of polypeptide transferred thereto by electroblotting, said hydrophobic layer being positioned beyond the hydrophilic membrane in a direction of movement of the fragments of polypeptide (usually cathode to anode); PA0 c) electroblotting the polypeptides from the separation gel through the hydrophilic membrane or membranes, under conditions effective to cause the polypeptides to be cleaved into fragments by the cleaving reagent, to the hydrophobic layer; and PA0 d) identifying the fragments collected on the hydrophobic layer and from the identification of the fragments, identifying the polypeptide from which they came. PA0 a) at least one hydrophilic membrane on which is immobilised at least one reagent capable of cleaving a polypeptide; and PA0 b) a hydrophobic collection layer suitable for receiving thereon fragments of polypeptide transferred thereto by electroblotting.
These types of processes are not practical for the sequencing of polypeptides which have been run on the same gel, since the cutting out of the polypeptide bands from the gel has to be done sequentially and the plugs thus obtained placed in tubes for further analysis. Also, losses occur when the polypeptides adhere to the walls of the tube.